Chemokines are small soluble proteins that stimulate chemotactic cell migration via activation of a G protein-coupled receptor (GPCR). In addition to their vital roles in inflammation, wound healing, and stem cell homing, chemokines also contribute to many pathologies including autoimmune diseases and cancer. Interactions of the chemokine CXCL12 (stromal cell-derived factor-1/SDF-1) and its receptor CXCR4 are particularly well studied because of their participation in neurogenesis, cardiogenesis, angiogenesis, myocardial infarction/reperfusion injury, HIV infection, and numerous carcinomas and sarcomas.
Chemokine receptor recognition and activation occurs via a two-step, two-site process. First, the CXCR4 extracellular N-terminus binds to CXCL12 (site 1). The N-terminus of CXCL12 then recognizes the receptor transmembrane domain and activates signaling (site 2). In addition to one site of O-linked glycosylation, CXCR4 possesses three tyrosine residues (Tyr7, Tyr12, and Tyr21) in the N-terminus capable of being O-sulfated in the Golgi apparatus. Mutational studies suggested that sulfation of Tyr21 enhances the binding of wild type CXCL12 (CXCL12WT, SEQ ID NO:1), but the level of sulfation at each tyrosine and their relative contributions to chemokine recognition were not quantified. Consistent with studies of the full-length CXCR4 receptor expressed in cells, sequential sulfation of a peptide comprising residues 1-38 (CXCR41-38) enhanced the affinity for CXCL12. Full-length chemokine receptors CCR2b, CCR5, CCR8, CXCR3, and CX3CR1 have since been shown to also exhibit increased ligand binding affinity upon tyrosine sulfation; replacement of tyrosines with phenylalanine residues resulted in 10 to 200-fold decreases in affinity and, in some cases, undetectable binding demonstrating the importance of sulfotyrosine recognition in chemokine signaling.
While most chemokines form dimers or other oligomers, receptor activation is typically restricted to the monomeric ligand. However, structure-function studies of CXCL12 using preferentially monomeric (CXCL12H25R) and constitutively dimeric (CXCL122) variants demonstrated that dimerization converts CXCL12 into a partial CXCR4 agonist that potently inhibits chemotaxis. As a CXCR4 ligand that stimulates intracellular calcium flux but fails to activate F-actin polymerization or β-arrestin recruitment, the CXCL122 dimer causes a type of ‘cellular idling’ that can block metastatic tumor formation in animal models for colorectal cancer and melanoma. Differences in how the CXCR4 N-terminus is recognized by CXCL12 monomers and dimers may contribute to their distinct receptor activation profiles (26). For instance, in the NMR structure of the CXCL122:CXCR41-38 complex the receptor fragment wraps around both subunits of the CXCL12 dimer, placing sulfotyrosine 12 (sTyr12) and sTyr21 in distinct sites on one subunit while sTyr7 occupies a cleft at the dimer interface that would not exist in a CXCL12 monomer. In concordance with this structural model, it was observed that CXCR41-38 binding promotes CXCL12 dimerization. However, individual contributions of CXCR4 sulfotyrosines to the affinity and specificity of CXCL12 recognition and their impact on the monomer-dimer equilibrium remain unknown.
Site 1 contacts that contribute most to binding are potential targets for development of novel chemokine probes and antagonists. For example, we recently demonstrated that the sTyr21 binding pocket of CXCL12 can be targeted for inhibition by small molecule ligands that block CXCR4-mediated calcium signaling and chemotaxis (29, 30). This appears to be a conserved binding site, suggesting that sulfotyrosine-guided drug discovery may be a general strategy for targeting the chemokine family and other protein-protein interactions in the extracellular space.
Accordingly, there is a current need for cost-effective pharmaceutical agents and treatment methods for treating various conditions including autoimmune or inflammation disorders, immune suppression conditions, infections, blood cell deficiencies, cancers and other described conditions and to mobilize stem cells by manipulating and controlling CXCL12 and CXCR4.